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Ligand induced changes in amino acid accessibility of P-Glycoprotein

University of British Columbia

P-Glycoprotein (Pgp) is a 170 kDa plasma membrane protein belonging to the ATP-Binding Cassette (ABC) superfamily. The expression of this protein in tumor cells leads to the emergence of a multidrug resistance phenotype (MDR) which is thought to be responsible for the failure of chemotherapy in some forms of cancer. Pgp is an ATP-dependent drug efflux pump, using the energy of ATP hydrolysis to mediate drug transport. The mechanism of substrate recognition and transport, however, is poorly understood. There is strong evidence that Pgp proceeds through various structural changes as it goes through its catalytic cycle. In the present study, non-selective fluorescent labeling with a residue-specific fluorescent modification agent was used to locate regions on Pgp that undergo conformational changes with substrate binding. A lysine-specific modification agent, DEACSE, was used to label Pgp and fluorescent peptide maps were generated by protease cleavage. The labeling profiles of Pgp in the presence of various substrates were compared to pinpoint regions that exhibited ligand-dependent changes in accessibility to DEACSE. Several cleavage products show significant changes in fluorescence intensity with different substrates. Selected peptides were identified by MALDI-TOF mass analysis and N-terminal peptide sequencing. Two peptides with reduced labeling upon nucleotide binding were localized respectively to the amino (525GAQLSGGQKQR) and carboxyl (1170GTQLSGGQKQR) Walker C signature motif within the nucleotide binding domains of Pgp (modified lysine residue in bold and underlined). On the other hand, two peptides with increased labeling were observed in the predicted second cytoplasmic loop. Peptide 260TVIAFGGQKK appears to be the most dynamic and experienced a significant increase in fluorescence while the second peptide, 284LGIKK, encountered a more modest increase. These findings indicate that the non-selective fluorescent modification technique developed in this study can be used to map in greater detail areas of conformational changes in Pgp. This technique should be applicable also to other protein systems, including a number of ABC proteins. Initial results from this study are consistent with the proposal by others that the Walker C signature sequence may be involved in coupling ATP binding and/or hydrolysis to substrate transport. In addition, they show that nucleotide binding can induce significant structural changes within the transmembrane domains located at a distance along the linear sequence of the protein from the nucleotide binding sites. These results provide evidence that supports a mechanism in which energy is transduced to effect a global conformational change associated with transport. [Certain scientific formulae used in this abstract could not be reproduced.]

Thesis/Dissertation

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2009-05-23

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http://hdl.handle.net/2429/8089

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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